This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acidic glycosylation patterns such as sialylation, phosphorylation and sulfation are known to change in recombinant glycoproteins under different cell culture conditions. Phosphorylated high mannose glycans are particularly important for targeting lysosomal disorders with enzyme replacement therapies. While less is known about the effects of sulfated N-linked glycans, this carbohydrate modification has been implicated in receptor-mediated uptake and protein clearance. Characterizing and monitoring this glycan modification is important for maintaining a robust and efficacious therapeutic product. The analytical challenges of characterizing sulfated N-linked glycans arise from distinguishing the sulfation from phosphorylation, retaining this labile modification, and obtaining a robust mass spectrometry-compatible purification. The present study demonstrates the challenges and strategies toward characterizing sulfated complex glycans on large glycoproteins. Enrichment of protein species with sulfated glycans was beneficial for detecting low levels of sulfation and was accomplished through strong anion exchange. Glycopeptides with a single glycosylation site were obtained by a combined LysC/GluC proteolytic digestion of lysosomal enzymes over 24 hours. LC/MS glycopeptide characterization was performed on an LTQ Orbitrap mass spectrometer with a prior reverse-phase separation. Multiple glycopeptide fractions were collected through automated fraction collection. Subsequent release of site-specific glycans was performed by PNGase F digestion for 1 hour at 37 [unreadable]C and further purified from the remaining peptides through solid-phase extraction. HILIC chromatography was used to characterize the acidic glycans on an Agilent 6520 Q-TOF interfaced with a chip cube source.